Project Summary/Abstract The current level of research activity involving gene therapy with either synthetic vectors (carriers) or engineered viruses is unprecedented. Liposomes are the most widely studied nonviral carriers worldwide for nucleic acid (NA) and drug delivery applications. Cationic liposomes (CLs) are relatively safe nonviral vectors used in ongoing clinical trials. CLs may either be complexed via electrostatic interactions with therapeutic NAs (anionic DNA or short interfering RNA) for gene delivery and silencing, or used as vectors of potent cytotoxic hydrophobic drugs, encapsulated within their lipid bilayer, in cancer therapeutics. Among the biggest advantages of nonviral vectors (over viral vectors which are currently more efficient in in vivo settings) are their safety, their low immunogenicity and their ability to transfer entire genes (containing coding and noncoding sequences) and regulatory sequences into cells (currently not feasible with engineered viruses because of capsid size limitations). The development of nonviral lipid-based vectors with efficacy competitive with viral vectors in vivo will require a mechanistic understanding of how synthetic vectors may be functionalized to overcome the major intracellular hurdle of endosomal escape. Successful endosomal escape is required for release of therapeutic nucleic acid within the cell cytosol and therefore maximum efficacy. The first aim of this research application is to employ modern biophysical and synthetic approaches to the rational design of functionalized CL?NA nanoparticles (NPs) with synergistic, complementary dual-function PEG-lipid and fusogenic components for optimized endosomal escape. Modern methods of organic and solid phase chemistry will be employed to synthesize dual-function PEG-lipids with cell targeting and endosome escaping properties. The second aim of this research application is to optimize efficacy of a new class of CL-based carriers of the hydrophobic drug paclitaxel (PTXL) for cancer therapeutics. This will be achieved by developing a mechanistic understanding of the relation between physical and chemical properties of the carrier (i.e. size of the functionalized CL carrier, membrane spontaneous curvature, and lipid tail structure) and functional efficacy (i.e. PTXL membrane solubility, cell uptake of vector and PTXL delivery leading to cytotoxicity against human cancer cells). The structures of CL-based vectors of NAs and hydrophobic drugs will be characterized using cryogenic electron microscopy and synchrotron x-ray diffraction techniques. The interactions between CL vectors and cell organelles will be directly visualized with spinning disk confocal fluorescence microscopy. Their structures will be correlated to their biological activity in human cancer cells. The broad, long-term objective of our research is to develop a fundamental science base through mechanistic studies that will lead to the design and synthesis of nonviral vectors of nucleic acids and hydrophobic drugs for gene and cancer therapeutics.